Genes that encode proteins essential for microbial growth or viability are useful targets for antibiotics because inhibition of such proteins by an antibiotic can reduce or eliminate the spread of infectious disease in a patient. The emergence of bacteria resistant to multiple antibiotics has led to renewed interest in isolating variants of known antibiotics and also in identifying new required genes and the corresponding gene products that could serve as new targets for novel antibiotics.
Approximately fifteen different bacterial proteins encoded by essential genes have been used as targets for antibiotics. Such target proteins include ribosomal proteins, gyrase, RNA polymerase and proteins involved in the synthesis of the peptidoglycan layer and its precursors. However, there are estimated to be more than 4000 putative genes in the genome of the bacterium Escherichia coli and it is not known how many of these genes are required for growth and/or viability. The discovery of additional required genes could facilitate the search for new antibiotics.
Microbial genes required or essential for growth and/or viability have been identified by two major techniques. One approach is to determine the nucleotide sequence of the genome of a microbial species of interest. Sequence information is then compared to sequences in computer databases to identify possible functions for the putative gene sequences. To prove that a putative gene identified only by sequence comparisons is in fact an essential gene, however, a null or “knockout” mutation must be made in the gene. It must be shown that a microorganism containing the null mutation cannot survive unless it is complemented by a wild type allele of the gene. Such an approach is time consuming and, in some species, can be difficult.
A second approach is to isolate conditionally lethal mutants by means such as chemical mutagenesis. A common type of conditional lethal mutation is a temperature-sensitive mutation, in which the mutant is non-viable at higher temperature such as 42° C. but is viable at a lower temperature such as 30° C. The mutation is then mapped and identified by cloning and genetic complementation. The mutagenesis techniques employed are sometimes not completely random, thus reducing the likelihood of identifying a required gene that does not have a “hot spot” for the mutagen used. Moreover, products of some required genes may not be amenable to the formation of conditional lethal mutants. Such essential genes would not be identified by this approach.